Biomedical subjects

D A Mickle

Publications and source records attributed to D A Mickle.

At least 19 recordsLinked to original sources

PubMed1996

Does the internal thoracic artery graft delay the recovery of myocardial metabolism?

BACKGROUND: The left internal thoracic artery (LITA) bypass graft to the left anterior descending artery has greater long-term patency than a saphenous vein graft. However, surgeons may be reluctant to use the LITA graft in some patients because they are unable to deliver cardioplegia to the left anterior descending artery territory. METHODS: We compared the myocardial levels of high-energy phosphates and their metabolites in patients who received an LITA graft with those in patients who received a saphenous vein graft to the left anterior descending artery territory during elective coronary artery bypass grafting. Right and left ventricular biopsy specimens were obtained at three times: before aortic cross-clamping, after cross-clamp removal, and after 10 minutes of reperfusion. RESULTS: No differences were found between the LITA graft group and the saphenous vein graft group in any right ventricular metabolites. There was an improvement in myocardial protection over time and a higher proportion of LITA graft patients in the late time period (early group, 63% versus late group, 80%; p < 0.01). Within each time period, there were no differences between the LITA and saphenous vein graft groups. Among patients receiving cold antegrade cardioplegia, the myocardial levels of high-energy phosphates were better preserved in those receiving an LITA graft. CONCLUSIONS: Advances in myocardial protection have led to improved preservation of high-energy phosphate levels after cardioplegic arrest. In patients undergoing elective coronary artery bypass grafting, the use of an LITA graft does not adversely affect myocardial metabolism. Further investigations are required to determine the effects of the use of the LITA during urgent or emergent procedures.

Adenine Nucleotides↗

PubMed1996

Human pediatric and adult ventricular cardiomyocytes in culture: assessment of phenotypic changes with passaging.

OBJECTIVES: The purpose of this study was to assess morphologically and biochemically the phenotypic changes which occur in vitro with passaging of human pediatric and adult ventricular cardiomyocytes. METHODS: Human ventricular cardiomyocytes from 3 children (1 to 2 years of age) and an adult patient (65 years of age) undergoing open heart surgery and an adult heart transplant patient (55 years of age) were isolated, cultured, purified, and passaged. Growth curves and 3H-thymidine uptake studies were performed. Characterization of the cells was done by light microscopy, transmission electron microscopy, immunofluorescent staining for myoglobin, CK-MB, and cardiac-specific troponin I isoform, human ventricular myosin heavy chain (HVMHC) and light chain 1 (HVMLC1), Northern blot analysis of HVMHC, and CK-MB activity and mass measurements. Passage 3 cardiomyocyte and pediatric myocardial phospholipids were analysed by gas chromatography. RESULTS: Pediatric cells were smaller (P < 0.01) and divided faster (P < 0.001, ANOCOVA) than adult cells. The cardiomyocytes showed phenotypic changes in primary culture with essentially complete loss of sarcomeres by 10 days and a gradual loss of myofilaments with passaging. The cells were identified as cardiomyocytes by immunohistochemistry for myoglobin, CK-MB, cardiac-specific troponin I isoform, HVMHC and HVMLC1, and by Northern blot analysis for the 3'-end of HVMHC mRNA. The composition of phospholipid fatty acids in the cultured pediatric cells was similar to that found in the pediatric myocardium. CK-MB activity and mass could be measured in the cardiomyocytes. The adult cardiomyocytes were more difficult to maintain than the pediatric cells which could be cultured for as long as 6 months. CONCLUSIONS: Primary cultures of human pediatric and adult partially differentiated ventricular cardiomyocytes can be passaged. Although rapid disorganization of the myofibrils occurs, the non-contractile cells can be identified as cardiomyocytes by morphological appearance, immunofluorescent staining, Northern blot analysis for HVMHC, and CK-MB activity.

Actin Cytoskeleton↗

PubMed1996

Effect of vitamin E on human glutathione peroxidase (GSH-PX1) expression in cardiomyocytes.

To determine the effect of vitamin E on cellular antioxidant enzymes, human ventricular cardiomyocytes were incubated with 200 microM all-racemic-alpha-tocopheryl acetate for 14 d at pO2s of 150 and 40 mm Hg. Cellular Cu, Zn superoxide dismutase, catalase, and GSH-Px1 activities were measured. Although SOD and catalase activities were unaffected by alpha-tocopherol, GSH-Px1 activities increased (p < .0001) as much as twofold. This increase was independent of oxygen tension and selenium. The increase in GSH-Px1 activity became significant (p < .01) by day 4. A nonantioxidant analog of alpha-tocopherol, 200 microM RRR-alpha-tocopherol methyl ether, did not affect GSH-Px1 activities. Although GSH-Px1 mRNA levels mirrored the changes in enzyme activities, the de novo nuclear GSH-Px1 transcript synthesis was unaffected by alpha-tocopherol. Because the increase in GSH-Px1 activities also occurred after cellular alpha-tocopherol levels had plateaued, the above results were most consistent with posttranscriptional stabilization of GSH-Px1 mRNA by alpha-tocopherol or an alpha-tocopherol-related metabolic product.

Analysis of Variance↗

PubMed1996

Cardiomyocyte transplantation improves heart function.

BACKGROUND: Transplantation of cultured cardiomyocytes into myocardial scar tissue may prevent heart failure. METHODS: Scar tissue was produced in the left ventricular free wall of 15 rats (weight, 450 g) by cryoinjury. Seven animals had operation only and survived for 8 weeks (sham group). Four weeks after cryoinjury, cultured fetal rat cardiomyocytes or culture medium was injected into the scar tissue of transplantation (n = 5) and control (n = 5) animals, respectively. Five other rats were sacrificed for scar assessment. Eight weeks after cryoinjury heart function in the transplantation, control, and sham groups was measured using a Langendorff preparation. Histologic studies were performed to quantify the extent of the scar and the transplanted cells. RESULTS: Four weeks after cryoinjury, 36% +/- 4% (mean +/- 1 standard error) of the left ventricular free wall surface area was scar tissue. At 8 weeks, the scar size had increased (p < 0.01) to 55% +/- 3% in the control group. Although the scar size (43% +/- 2%) in the transplantation group at 8 weeks was not significantly different from that at 4 weeks, it was less (p < 0.05) than that in the control group. Hearts in the sham group had no scar tissue. The transplanted cardiomyocytes had formed cardiac tissue within the myocardial scar. Systolic and developed pressures in the transplantation group hearts were greater (p = 0.0001) than in the control group hearts but less (p < 0.01) than those in the sham group hearts. CONCLUSIONS: The transplanted cardiomyocytes formed cardiac tissue in the myocardial scar, limited scar expansion, and improved heart function compared with findings in the control hearts.

Animals↗

PubMed1996

Antegrade and retrograde cardioplegia: alternate or simultaneous?

UNLABELLED: Neither antegrade nor retrograde cardioplegic protection provides homogeneous distribution, and a combination may be required to avoid anaerobic metabolism and depressed postoperative ventricular function. Tepid cardioplegia (29 degrees C) avoids the delayed recovery of cardiac function and metabolism associated with cold cardioplegia (15 degrees C) and reduces the anaerobic metabolism seen with warm (37 degrees C) cardioplegia. We compared two techniques that combine antegrade and retrograde tepid cardioplegia: alternate and simultaneous. METHODS: Sixty patients undergoing elective isolated coronary artery bypass grafting were randomized to receive near continuous tepid retrograde and either intermittent antegrade cardioplegia (the alternate technique) or antegrade cardioplegia with the solution delivered concurrently through each completed vein graft (the simultaneous technique). RESULTS: Myocardial lactate extraction was greater after crossclamp release following simultaneous than alternate cardioplegia. Postoperative ventricular function was better after alternate than simultaneous cardioplegia. CONCLUSION: Both techniques permitted rapid postoperative recovery of myocardial metabolism and ventricular function. However, simultaneous cardioplegia was simpler and did not require deairing the aortic root between antegrade infusions.

Adult↗

PubMed1996

In vivo survival and function of transplanted rat cardiomyocytes.

Recent studies have demonstrated the feasibility of transplanting fetal mouse cardiomyocytes into the hearts of adult syngeneic mice. However, the function of the transplanted cardiomyocytes and their capacity to survive in fibrous connective tissue were not assessed. In the present study, we evaluated the viability and contractility of transplanted fetal and neonatal rat cardiomyocytes in the connective tissue of the adult rat hindlimb. Purified fetal or neonatal rat cardiomyocytes were cultured. These cells contained sarcomeres, formed junctions composed of desmosomes and fascia adherens, and contracted regularly and spontaneously. A fetal or neonatal cardiomyocyte suspension was injected into the subcutaneous tissue of adult rat hindlimbs. Cyclosporin A (5 mg/kg) was administered subcutaneously daily for the 3-month duration of the study, at which time the animals were killed. The transplanted cardiomyocytes formed 'tissue' in vivo that increased in size for the first 2 weeks and remained the same size at the third week. The tissue derived from the transplanted fetal cardiomyocytes contracted spontaneously at a rate of 73 +/- 12 bpm, and that from the neonatal cardiomyocytes contracted at a rate of 43 +/- 21 bpm. The electrocardiogram was similar to that seen in myocardium with an idioventricular rhythm. Histologically, the tissue appeared to be cardiac muscle with sarcomeres. Angiogenesis occurred in the cardiomyocyte graft. In summary, a cell suspension of cultured fetal and neonatal rat cardiomyocytes transplanted into the adult rat hindlimb formed contractile cardiac tissue in the subcutaneous connective tissue.

Animals↗

PubMed1995

Tepid antegrade and retrograde cardioplegia.

To determine the optimal temperature for the combination of antegrade and retrograde cardioplegia, 42 patients undergoing coronary artery bypass grafting were randomized to receive cold (9 degrees C; n = 14), tepid (29 degrees C; n = 14), or warm (37 degrees C; n = 14) blood cardioplegia delivered continuously retrograde and intermittently antegrade. Myocardial oxygen utilization, lactate and acid metabolism, and coronary vascular resistance were measured during the operation and cardiac function was assessed postoperatively. Myocardial oxygen consumption, lactate release and acid release were greatest with warm, intermediate with tepid, and least with cold cardioplegia (p = 0.0001). However, washout of lactate and acid at the time of cross-clamp release was reduced (p = 0.022) with tepid or cold compared with warm cardioplegia. Early postoperative left ventricular function was best preserved (p = 0.01) after tepid than after cold or warm combination cardioplegia. These results suggest that tepid combination cardioplegia reduced metabolic demands but permitted immediate recovery of cardiac function. This technique may provide better myocardial protection than cold or warm combination cardioplegia.

Aged↗

PubMed1995

Cardiac storage with University of Wisconsin solution and a nucleoside-transport blocker.

Findings from previous investigations conducted at this institution and others have suggested that University of Wisconsin solution (UWS) is preferable for the prolonged hypothermic storage of hearts before transplantation. The benefit seen with UWS may in part be related to the inclusion of adenosine (5 mmol/L) in the UWS. To investigate whether further manipulations of adenosine metabolism might enhance myocardial protection, studies were initially conducted using cultured myocytes, followed by confirmatory experiments using isolated rat hearts. Cultured human ventricular myocytes (7 to 8 dishes/group) were stored for 12 hours at 0 degrees C in unmodified UWS or UWS supplemented with increasing concentrations (1 to 100 mumol/L) of the nucleoside-transport blocker p-nitrobenzylthioinosine. The adenosine triphosphate concentrations were found to be enhanced with nucleoside-transport inhibition, with the best results achieved with the 1- and 3-mumol/L groups (control, 3.37 +/- 0.41 nmol/micrograms DNA; UWS, 2.89 +/- 1.31 nmol/micrograms DNA; 1 mumol/L, 5.91 +/- 3.23 nmol/micrograms DNA; 3 mumol/L, 7.86 +/- 3.45 nmol/micrograms DNA; p < 0.05 versus control or UWS group). Isolated rodent hearts from Sprague-Dawley rats were prepared on a Langendorff apparatus with an intraventricular balloon and subsequently stored for 8 hours at 0 degrees C in unmodified UWS (13 hearts/group) or UWS supplemented with 1 or 3 mumol/L of p-nitrobenzylthioinosine (9 to 10 hearts/group).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗

PubMed1995

Adequate distribution of warm cardioplegic solution.

Seventy-five patients undergoing coronary artery bypass grafting were randomized to receive warm antegrade (N = 25), warm retrograde (N = 25), or a combination of warm antegrade and retrograde (N = 25) delivery of blood cardioplegic solution. Myocardial oxygen utilization, lactate and acid metabolism, and adenine nucleotides and their degradation products were measured during the operation and cardiac function was assessed postoperatively. Warm retrograde delivery of cardioplegic solution increased lactate and acid release during cardioplegia and reperfusion, decreased left ventricular adenosine triphosphate concentrations, and reduced the washout of adenine nucleotide degradation products from both left and right ventricles. Warm antegrade delivery of cardioplegic solution resulted in less lactate and acid release during cardioplegia but more lactate accumulated in the territory of the left anterior descending artery during the crossclamp period. Intermittent antegrade delivery of the cardioplegic solution during combination cardioplegia washed out lactate and acid, which suggested inhomogeneous delivery of the cardioplegic solution during continuous retrograde cardioplegia. Combination cardioplegia best preserved adenosine triphosphate in the left ventricle and resulted in the best postoperative left and right ventricular function. A combination of intermittent antegrade and continuous retrograde delivery of cardioplegic solution provided better myocardial protection than either antegrade or retrograde delivery of cardioplegic solution alone.

Adenine Nucleotides↗

PubMed1995

Adenosine pretreatment for prolonged cardiac storage. An evaluation with St. Thomas' Hospital and University of Wisconsin solutions.

Adenosine pretreatment has been shown to be beneficial in several models of ischemia-reperfusion. We wished to evaluate whether adenosine pretreatment is cardioprotective for prolonged cardiac storage and whether the presence of adenosine in the storage media affects the results. Isolated rodent hearts were obtained from Sprague-Dawley rats, mounted on a Langendorff apparatus, instrumented with an intraventricular balloon, and ventricularly paced at 300 beats/min. Four groups of hearts were studied in a 2 x 2 factorial experiment (n = 8 to 12 per group). Hearts were subjected to normal perfusion or to solution supplemented with adenosine 50 mumol/L for 10 minutes followed by adenosine-free perfusion for 10 minutes. Hearts then were stored for 8 hours at 0 degrees C in either University of Wisconsin solution (adenosine 5 mmol/L) or St. Thomas' Hospital II solution (adenosine free). Adenosine pretreatment increased tissue levels of adenosine triphosphate before storage (p = 0.04). Nonfunction was less common after storage (1/19 versus 6/20 hearts, p < 0.05), and diastolic function was better preserved in the adenosine groups in the reperfusion phase (p = 0.01). The beneficial effects of adenosine pretreatment were independent of which storage solution was used. Developed pressure was increased (p < 0.05) and release of creatine kinase and lactate dehydrogenase was reduced (p < 0.0001) in hearts treated with University of Wisconsin solution compared with those treated with St. Thomas' Hospital solution. These studies suggest that adenosine pretreatment improves recovery after prolonged hypothermic storage and that the presence of adenosine in the preservation solution does not alter the results. The experiments provide further evidence that extended myocardial protection is better enhanced with University of Wisconsin solution than with St. Thomas' Hospital II solution.

Adenosine↗

PubMed1994

The optimal cardioplegic temperature.

Seventy-two patients undergoing coronary artery bypass grafting were randomized to receive cold (8 degrees C) antegrade or retrograde, tepid (29 degrees C) antegrade or retrograde, or warm (37 degrees C) antegrade or retrograde blood cardioplegia (n = 12 in each group). Myocardial oxygen utilization as well as lactate and acid metabolism were assessed intraoperatively and cardiac function was assessed postoperatively. Myocardial oxygen consumption and anaerobic lactate release were greatest during warm, intermediate during tepid, and least during cold cardioplegic arrest. Myocardial oxygen consumption and lactate release were underestimated during retrograde cardioplegia because of contamination of aortic root samples. Warm retrograde and tepid retrograde cardioplegia resulted in greater lactate and acid washout with reperfusion. Left ventricular stroke work indices were greater after warm antegrade and tepid antegrade cardioplegia than after cold antegrade cardioplegia, and right ventricular stroke work indices were greatest after warm antegrade cardioplegia. Warm antegrade cardioplegia increased aerobic metabolism during and after cardioplegia and preserved left and right ventricular function. Tepid antegrade cardioplegia reduced anaerobic lactate and acid release during arrest and preserved cardiac function.

Adult↗

PubMed1994

Cardiac storage with UW solution and glucose.

Previous investigations from our institution using an isolated human cardiomyocyte model concluded that glucose supplementation of University of Wisconsin solution (UWS) was beneficial with respect to adenine nucleotide and protein recovery. We wished to confirm these results using an isolated heart model. Rodent hearts were frozen in liquid nitrogen (control) or flushed and stored in UWS for 8 hours at 0 degrees C or UWS supplemented with 10, 20, or 30 mmol/L glucose. Experimental hearts were assessed at end-storage or after 45 minutes of reperfusion on a Langendorff apparatus. Adenine nucleotides were assessed by high performance liquid chromatography. In parallel experiments, ventricular function was assessed before and after storage in Langendorff-perfused hearts instrumented with a left ventricular balloon. Glucose supplementation was associated with greater poststorage (20 and 30 mmol/L glucose) and postreperfusion (10, 20, and 30 mmol/L glucose) adenosine triphosphate levels than unmodified UWS. Developed pressure (expressed as a percentage of control values) was increased with 10 mmol/L glucose (75.2% +/- 7.9%, mean +/- standard deviation) compared with unmodified UWS (64.6% +/- 6.6%; p < 0.05). Coronary flow was greater with 10 (72.6% +/- 10.7%) or 20 mmol/L (71.2% +/- 12.5%) versus 0 mmol/L glucose (58.6% +/- 12.1%, p < 0.05). The data support previous in vitro findings and suggest that the addition of 10 mmol/L glucose to UWS is associated with enhanced recovery after prolonged hypothermic storage.

Adenine Nucleotides↗

PubMed1994

Preconditioning human ventricular cardiomyocytes with brief periods of simulated ischaemia.

OBJECTIVE: The aim was to test for "ischaemic" preconditioning in monolayer cultures of quiescent human ventricular cardiomyocytes. METHODS: Stabilised cardiomyocytes (n = 8 plates per group) were preconditioned with varying periods of simulated ischaemia and reperfusion, followed in all groups by 90 min of sustained "ischaemia" with or without 30 min of reperfusion. Cellular injury was assessed by trypan blue exclusion and survival was assessed by culturing the cells for 24 h postintervention. In addition, separate groups of cell plates (n = 8 per group) which had first been preconditioned with 20 min ischaemia and 20 min reperfusion were exposed to either 30, 60, or 90 min sustained ischaemia or 90 min sustained ischaemia with 30 min reperfusion. The supernatants and/or cell homogenates were analysed for hydrogen ion, lactate, lactate dehydrogenase (LDH), and adenine nucleotides and degradation products. RESULTS: Preconditioning (PC) decreased trypan blue uptake following subsequent sustained ischaemia, with the 20 min ischaemia/20 min reperfusion (20/20) regimen having the most profound effect [control ischaemia: 37.0(SEM 2.1); 10/10: 23.9(1.5); 20/20: 15.4(1.4); 30/30: 25.8(2.1) percent blue stained cells, p < 0.05 by ANOVA/Duncan]. The 20/20 preconditioning regimen resulted in less hydrogen ion [control: 2.1(0.4); PC: 1.4(0.1) mmol.g-1 protein, p < 0.05] and less LDH release [control: 20.7(3.1); PC: 11.9(4.2) units.g-1 protein, p < 0.05]. At 90 min of sustained ischaemia, the control group had produced significantly greater lactate [intracellular: control 1.55(0.62); PC 0.54(0.23) mol.g-1 DNA, p < 0.05; extracellular: control 0.47(0.09); PC 0.33(0.07) mol.g-1 DNA, p < 0.05]. There were no differences in ATP depletion in the two groups. CONCLUSIONS: Ischaemic preconditioning can be induced in human cardiomyocytes independent of other cell types. The effect can be established in human cell cultures.

Cells, Cultured↗

PubMed1994

Ischemic preconditioning: cardioprotection for cardiac surgery.

Traditionally, surgeons have attempted to minimize myocardial ischemic and reperfusion injury during cardiac procedures by optimizing cardioplegic solutions and modifying the conditions of reperfusion. New evidence suggests that in addition to these two strategies, surgeons may be able to induce myocardial resistance to ischemic injury, which permits immediate functional and metabolic recovery after cardiac operations. Although brief episodes of cardiac ischemia may be associated with mechanical and metabolic dysfunction ("stunning"), they have also been shown to protect against damage resulting from a subsequent prolonged ischemic episode. This phenomenon, known as ischemic preconditioning, has been extensively characterized since its original description in 1986. Recent studies in surgical models of cardioplegic arrest and reperfusion have suggested that the preconditioned, arrested heart may have an increased tolerance to prolonged ischemia and improved functional recovery after reperfusion. The development of a pharmacological agent that induces the preconditioning effect may revolutionize cardioprotection for cardiac surgery. We will review the characteristics of preconditioning and data supporting the application of this natural protective capacity to reduce ischemic damage during cardiac procedures.

Animals↗

PubMed1994

Oxyradical-induced antioxidant and lipid changes in cultured human cardiomyocytes.

Because the chronically cyanotic myocardium is thought to be more susceptible to oxyradical injury than the noncyanotic myocardium during cardiovascular surgery, we studied the oxyradical susceptibility of human ventricular cardiomyocytes cultured at high and low oxygen tension (PO2) levels. Passage 4 tetralogy of Fallot cardiomyocytes were cultured at PO2 levels of 150 and 40 mmHg for 14 days and then exposed for 10 min to superoxide radicals. The cellular levels of ATP, phospholipid fatty acids, phospholipid conjugated dienes, alpha-tocopherol, and activities of superoxide dismutase, catalase, and glutathione peroxidase were measured. ATP levels decreased more markedly in the low-PO2 group. Although the decrease in alpha-tocopherol levels was similar for both groups, phospholipid conjugated diene formation and phospholipid unsaturated fatty acid loss was greater in the low-PO2 cells. Glutathione peroxidase activity was rapidly inhibited. Superoxide dismutase activity was unaffected, and catalase activity was inhibited by no more than 50%. Although extracellular superoxide dismutase with catalase did not inhibit phospholipid conjugated diene formation and phospholipid breakdown, extracellular glutathione peroxidase with reduced glutathione did limit phospholipid damage. With the occurrence of membrane lipid peroxidation, the decreased glutathione peroxidase activity in the cyanotic tetralogy of Fallot myocardium determines cardiomyocyte membrane susceptibility to oxidant injury.

Cells, Cultured↗

PubMed1994

Vitamin E for coronary bypass operations. A prospective, double-blind, randomized trial.

BACKGROUND: Free radical lipid peroxidation contributes to the abnormal metabolism and ventricular function frequently seen after cardiac operations. Antioxidants may improve metabolic and functional recovery. METHODS: A prospective, randomized, double-blind clinical trial was conducted to determine the effects of vitamin E (alpha-tocopherol) (n = 14) or a corn oil placebo (n = 14) in patients undergoing elective coronary bypass operations. The RRR-alpha-tocopheryl acetate doubled the alpha-tocopherol levels in the heart. Myocardial metabolism and ventricular function were assessed after the operation. RESULTS: Atrial pacing induced myocardial lactate production in the control patients but lactate consumption in the alpha-tocopherol-treated patients on bypass 25 minutes after crossclamp release. Left ventricular stroke work indices were higher, at similar ventricular volumes, in the alpha-tocopherol-treated group, which indicates improved preload recruitable stroke work, and diastolic compliance was greater 4 hours after the operation. The postoperative creatine kinase cardiac isoenzyme levels were lower in the patients who received alpha-tocopherol. CONCLUSIONS: Pretreatment with alpha-tocopherol sufficient to double the myocardial concentrations had a small but significant metabolic and functional effect after elective coronary bypass operations when compared with placebo. These results do not justify pretreatment of low-risk patients, but they do justify an evaluation in high-risk patients.

Adult↗

PubMed1993

Identification of oxygen responsive elements in the 5'-flanking region of the human glutathione peroxidase gene.

The -1389 to +588 region of the human genomic glutathione peroxidase gene (hgpx1) was amplified using the polymerase chain reaction. This DNA fragment was cloned and sequenced, and various deletion constructs derived from the hgpx1 5'-flanking region were fused to the chloramphenicol acetyltransferase gene. These reporter genes were analyzed in transient transfection assays using primary cultured human ventricular cardiomyocytes obtained from patients with tetralogy of Fallot. Two distinct regions upstream from the transcription start site, which was determined using S1 nuclease analysis, were identified to be responsive to the oxygen tension in culture (pO2 values of 150 or 40 mm Hg). Methylation interference footprinting assays revealed proteins closely apposed to two sequences located at -1232 to -1213 and -282 to -275. We have designated these oxygen responsive elements ORE1 and ORE2, respectively. Gel mobility shift assays using double-stranded oligonucleotides corresponding to each site have demonstrated the formation of specific complexes using both cultured human cardiomyocyte and HeLa nuclear extracts. ORE1 and ORE2 bind disparate proteins with equal precision as bound complexes could be competed away with identical sequences but not with either the other ORE or an unrelated sequence. Insertion of these oxygen responsive elements into a reporter gene governed by a SV40 promoter similarly regulated chloramphenicol acetyltransferase activity according to the oxygen tension in culture.

Base Sequence↗

PubMed1993

Chemical syntheses of Trolox conjugates which protect human ventricular myocytes against in situ-generated oxyradicals.

Synthetic conjugates of the antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylic acid) have been prepared by coupling it with 1-ethyl-3-(3-dimethyl-amino-propyl) carbodiimide hydrochloride either to p-aminophenyl-beta-D-lactopyranoside, or to higher molecular weight ligands such as dextran and polylysine. Compared to Trolox and on a mole to mole basis, dextran-Trolox is almost equally active, while lactosylphenyl- and polylysine-Trolox conjugates are distinctly more active in preventing the damage on human ventricular myocytes by oxyradicals generated from xanthine oxidase-hypoxanthine. Listed in order of decreasing cytoprotective activity, they are: lactosylphenyl-Trolox >> polylysine-Trolox > Trolox > dextran-Trolox. Thus, Trolox can be chemically modified by coupling it to one of a number of ligands and, in some cases, with resultant increases in its ability to protect human ventricular myocytes from oxyradical damage.

Antioxidants↗